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Genes to Cells (2007) 12, 623-638. doi:10.1111/j.1365-2443.2007.01073.x
© 2007 Blackwell Publishing or its licensors
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Rho-kinase dependent organization of stress fibers and focal adhesions in cultured fibroblasts

 Kazuo Katoh*, Yumiko Kano and Shigeo Ookawara

Department of Anatomy, School of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-city, Tochigi 329-0498, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The activation of Rho-kinase is known to modulate the organization of the actin-based cytoskeletal systems, including the formation of stress fibers and focal adhesions. Rho-kinase likely plays a more crucial and complex role in the organization of actin-based cytoskeletal systems than in that of myosin light chain kinase (MLCK). In order to understand the role of Rho-kinase in the organization of stress fibers and focal adhesions, we treated cultured fibroblasts with a Rho-kinase inhibitor and analyzed the stress fiber and focal adhesion organization under conventional fluorescence microscopy and replica electron microscopy. Some of the cells were transfected with GFP-labeled paxillin, actin or {alpha}-actinin, and the effects of the inhibitor were monitored in the living cells. The Rho-kinase inhibitor caused disassembly of the stress fibers and focal adhesions in the central portion of the cell within 1 h. However, the stress fibers and focal adhesions located in the cell periphery were not as severely affected by the Rho-kinase inhibitor. The time-lapse video recording revealed that when these cells were washed with a fresh medium in order to remove the Rho-kinase inhibitor, the stress fibers and focal adhesions located in the center of the cell gradually reorganized and, within 1.5–2 h, the cells completely recovered. This observation strongly suggests that the activation of Rho-kinase plays an important role in the organization of the central stress fibers and focal adhesions.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Stress fibers are contractile apparatuses which consist of prominent bundles of actin and myosin filaments. Stress fibers terminate in the focal adhesions, in which several of the structural proteins that connect the plasma membrane to the extracellular matrix are localized. It is generally known that the contraction of non-skeletal muscle cells, including stress fibers and smooth muscle cells, is caused by the phosphorylation of myosin regulatory light chain (MLC) by a calmodulin/myosin light chain kinase (MLCK) system in a Ca2+-dependent manner.

Rho-associated kinase (called Rho-kinase, ROCK2 or Rok{alpha}) is an effector of Rho small GTPase which is involved in the signal transduction mechanisms of the cell. The activation of Rho-kinase is known to modulate the organization of both stress fibers and focal adhesions (Ridley & Hall 1992; Amano et al. 1996a; Totsukawa et al. 2000). Rho-kinase is known to be phosphorylated directly or indirectly by MLC in in vitro systems in a Ca2+-independent manner (Amano et al. 1996a,b; Totsukawa et al. 2000). Rho-kinase direct phosphorylation of MLC at serine-19 results in the contraction of the non-muscle cell systems (Amano et al. 1996a; Kureishi et al. 1997), such as stress fibers (Chihara et al. 1997; Katoh et al. 2001b). However, Rho kinase inhibits the myosin phosphatase activity through the phosphorylation of the myosin binding subunit (MBS) of myosin phosphatase (Kimura et al. 1996). The inhibition of myosin phosphatase activity increases the phosphorylation level of MLC, resulting in the contraction of the non-muscle actomyosin contractile systems.

The stress fibers that run in the basal portion of cultured cells can be broadly divided into two morphological types (Totsukawa et al. 2000; Katoh et al. 2001a): thick and dense stress fibers, which are located in the peripheral portion of the cell ("peripheral stress fibers"), and stress fibers, which are located in the central portion of the cell ("central stress fibers"). We and other groups have previously demonstrated that there are two independent control systems in the contraction of the stress fibers in the basal portion of the cell: (i) peripheral stress fibers which are more sensitive to an MLCK inhibitor than to a Rho-kinase inhibitor, and (ii) central stress fibers which are more sensitive to a Rho-kinase inhibitor than to an MLCK inhibitor (Totsukawa et al. 2000; Katoh et al. 2001a). We have also demonstrated, by using isolated stress fibers in fibroblastic cells, that the MLC phosphorylation induced by Rho-kinase alone is slow and weaker than that induced by MLCK (Katoh et al. 2001b). Moreover, this type of contraction is sustained and fine-tuned in living fibroblastic cells. The Rho-kinase-dependent contraction system seems to be suitable for maintaining a sophisticated contractile system within the cell.

{alpha}-Actinin is one of the major stress fiber associated proteins that plays a key role in bundling and maintaining the stability of the stress fibers (Byers et al. 1984; Burridge et al. 1988). In addition to the bundling function within the stress fibers, {alpha}-actinin also functions directly by connecting the stress fibers to focal adhesions via a focal adhesion structural protein named vinculin (Otto 1983). In vitro experiments have demonstrated that {alpha}-actinin directly binds to a transmembrane protein called integrins (Otey et al. 1990). Thus, {alpha}-actinin seems to play a key role in the organization of both stress fibers and focal adhesions.

In the basal portion of cultured cells, there are at least three types of cell-to-cell attachment sites: (i) typical focal adhesions which associate with both ends of the stress fibers, (ii) small adhesion plaque-like structures which localize periodically along the stress fibers (also called "fibrillar plaques"), and (iii) small and punctuated focal adhesion complexes which are located at the leading edge of the cell. The organization of the fibrillar plaques remains unclear. However, it is well known that the organization of the focal adhesion and the focal adhesion complex is controlled by Rho A and Rac, respectively (Nobes & Hall 1995). The focal adhesion complex is associated with the MLCK inhibitor but not with the Rho-kinase inhibitor, thus reflecting that the organization of the focal adhesion complex and the typical focal adhesion is independently regulated (Katoh et al. 2001a).

Although the regulation of Rho-kinase activity is well characterized in in vitro analysis, little is currently known about the regulation of Rho-kinase-dependent organization of stress fibers and focal adhesions. In addition, it still remains unknown regarding Rho-kinase-dependent stress fiber and focal adhesion organization occurs in living cells. In this report, we examine the Rho-kinase dependent organization of the stress fibers and focal adhesions in living fibroblasts using Rho-kinase-specific inhibitors. We demonstrate that (i) Rho-kinase specifically regulates the organization of the stress fibers and focal adhesions that are located in the central portion of the cell, and (ii) during the Rho-kinase-dependent organization of the focal adhesions, the focal adhesion associated protein is first accumulated in small plaque-like structures, followed by the accumulation of bundled actin filaments along these plaques. We therefore suggest that the organization and maintenance of the stress fibers and focal adhesions in the central portion of the adhered cell is largely dependent on the Rho-kinase activity.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Effects of Rho-kinase inhibitors on peripheral and central stress fibers

Stress fibers are the rod-shaped structures that are made by the actomyosin-based contractile systems in several types of cells. Our previous report suggested that there are two main types of stress fibers in the basal portion of the cultured cells: stress fibers that are located in the peripheral portion of the cell (peripheral stress fibers) and others that are located in the central portion of the cell (central stress fibers). Interestingly, the central stress fibers are more susceptible to the Rho-kinase inhibitors than to the MLCK/calmodulin inhibitors (Totsukawa et al. 2000; Katoh et al. 2001a). In order to observe the detailed stress fiber structures in both the peripheral and central regions at the electron microscopic level, we performed replica electron microscopy. Figure 1a shows a typical replica electron microscopy of both the peripheral and central stress fibers in a cultured fibroblast. Both the peripheral and central stress fibers were easily distinguishable on a glass surface. Thick and long microfilament bundles are observed in the peripheral portion of the cell (Fig. 1a; arrows). Several thin and short microfilament bundles are visible in the central portion of the cell (Fig. 1a; arrowheads). In the basal portion of the cell, before treatment with the Rho-kinase inhibitors, the well-developed peripheral and central stress fibers were easily distinguishable by replica electron microscopy. After treatment with Y-27632 it also revealed the bundles of microfilaments at the cell periphery to decrease but they were hardly affected even if they were treated with an inhibitor for 1 h (Fig. 1b; arrows). On the other hand, the bundles of microfilaments in the central portion of the cell disappeared and only loosely arranged, uni-directional microfilaments were visible (Fig. 1c). This finding demonstrates the fact that the central stress fiber bundling came unlaced after the treatment with the Rho-kinase inhibitor. In order to identify the stress fibers and focal adhesions in the fibroblasts, the Y-27632 or HA1077 treated fibroblasts were stained simultaneously with rhodamine-labeled phalloidin for the visualization of actin filaments and with anti-vinculin for the visualization of focal adhesions. The fibroblasts were then observed under conventional fluorescence microscopy (Fig. 1d–f). After incubation with Y-27632 for 1 h, the focal adhesions which were revealed by anti-vinculin at the cell periphery were reduced in size, but they remained intact (Fig. 1d). Interestingly, the focal adhesions in the central portion of the cell were greatly reduced in number (Fig. 1f). Only tiny clusters were independently localized with phalloidin stained filamentous structures (Fig. 1e for actin filament and f for arrowheads for vinculin). The treatment of HA1077, another potent Rho-kinase inhibitor, also showed similar results (data not shown). Detailed observation with replica electron microscopy after incubation with the inhibitor revealed that the microfilaments were arranged in a uni-directional formation without the bundles in the center of the cell (Fig. 1c), reflecting that the phalloidin positive actin filament distribution existed as a loosely arranged actin filament "carpet" in the center of the cell (Fig. 1e).


Figure 1
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  		Figure 1  The replica electron microscopy of the stress fibers isolated on a coverslip and the conventional fluorescence microscopy after the incubation with the Rho-kinase inhibitor, Y-27632. The peripheral stress fibers (a; arrows) and the central stress fibers (a; arrowheads) were clearly distinguishable before the incubation with Y-27632. After incubation with Y-27632 for 1 h, the tight bundles of microfilaments were still present in the cell periphery (b; arrows), but the loose and uni-directional bundles of microfilaments were observed in the central portion of the cell (c). Conventional fluorescence microscopy after the incubation with Y-27632 for 1 h is shown in (d)–(f). The cells that were incubated with Y-27632 for 1 h were fixed and then doubly stained with rhodamine-labeled phalloidin (e) and anti-vinculin (f). The yellow staining in (d) indicates overlapping staining of the two colors. After the Y-27632 treatment, the cells had peripheral stress fibers (e) but only small focal adhesion-like structures in the cell center of the cell (f; arrowheads). Bar in (a)–(c) is 1 µm. Bar in (f) is 20 µm.

 
{alpha}-Actinin distribution during Rho-kinase-dependent reorganization of stress fibers and focal adhesions

It is widely known that the actin filaments in cells are organized in stress fiber bundles using actin bundling proteins called {alpha}-actinin. It is also known that {alpha}-actinin directly connects the actin filaments and focal adhesion associated proteins. {alpha}-Actinin thus seems to play an important role in bundling the actin filaments and in organizing focal adhesions. The actin filament organization during Rho-kinase-dependent stress fiber formation has already been examined by our and other groups (Totsukawa et al. 2000; Katoh et al. 2001a,b). However, the detailed {alpha}-actinin distribution during the Rho-kinase-dependent organization of the stress fibers and focal adhesions has not yet been examined. The effects of the Rho-kinase inhibitor on the stress fibers and focal adhesions were examined using living cells which were transfected with GFP-{alpha}-actinin. The disruption of the central stress fibers by the incubation with Y-27632 was recorded under conventional inverted fluorescence microscopy (Fig. 2). The time-lapse fluorescence microscopy revealed that the GFP-{alpha}-actinin transfected cells had stress fibers and focal adhesions which were clearly visible before the inhibitor was added (Fig. 2a; 0 : 00 min. Focal adhesions and stress fibers are indicated by arrows; see Supplementary Video S1 for Quicktime movie). When the inhibitor was added, the central stress fibers gradually disappeared within 20 min, although some of the GFP-{alpha}-actinin positive focal adhesion-like structures were still visible in the center of the cell (Fig. 2a; arrow in 0 : 30 and 0 : 60 min). After incubation with the inhibitor, the newly organized lamellopodium in the cell periphery was observed (Fig. 2a; 0 : 30 min, arrowheads). Since the expressed GFP-{alpha}-actinin gene products tend to generate a significant level of background fluorescence, we used total reflection fluorescence microscopy (TIRFM) to reduce background noise. The TIRFM image also revealed that, after the incubation with the inhibitor, the central stress fibers disappeared. However, some of the focal adhesions remained in the central portion of the cell (Fig. 2b; see Supplementary Video S2). There were fewer focal adhesions revealed by the GFP-{alpha}-actinin transfected cells than those revealed by immuno-staining with anti-vinculin antibodies. Both the conventional fluorescence and TIRFM images did not reveal all of the focal adhesion spots in the GFP-{alpha}-actinin transfected cells. This discrepancy was caused by the accessibility of GFP-{alpha}-actinin, which connects only stress fibers to vinculin and integrins. If the connection between the stress fibers and focal adhesion was significantly small, the accessibility of GFP-{alpha}-actinin would then be confined to a limited area. Some of the small focal adhesion-like structures could not be distinguished from the GFP-{alpha}-actinin transfected cells. The TIRFM image revealed that the typical {alpha}-actinin banding pattern on the stress fibers largely disappeared within 15–20 min (Fig. 2b; 0 : 15 min). However, many spotty {alpha}-actinin patterns were found in the center of the cell after the treatment of Y-27632 for 1 h (Fig. 2c; 0 : 60 min). As the TIRFM image selectively revealed a limited area from the glass surface, the above findings indicate that many of the {alpha}-actinin cells were still localized on the plasma membrane in a dot-like configuration after the incubation with the inhibitor.


Figure 2
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  		Figure 2  The effects of the Rho-kinase inhibitor in living fibroblasts expressing GFP-labeled {alpha}-actinin. Y-27632 was added at time 0 : 00, and the cells were imaged by time-lapse fluorescence microscopy (a). Note that the central stress fibers were gradually loosened, while the lamellopodium located at the cell periphery were formed in the presence of the Rho-kinase inhibitor (a; 0 : 30, arrowheads). See Supplementary Video S1. A TRIFM time-lapse image of the living fibroblasts expressing GFP-{alpha}-actinin treated with Y-27632 for 1 h was indicated in (b). See Supplementary Video S2. Y-27632 was added at time 0 : 00, and the cells were imaged by TRIFM. Note that the central stress fibers gradually disappeared over time. However, some of the focal adhesion-like structures remained unchanged. The insets in (b; 0 : 00 and 0 : 15) indicate the high-power view of the boxed area. Some dot-like patterns were also visible on the plasma membrane (Insets in b, 0 : 00 and 0 : 15). The recovery of the stress fibers in the living cell was indicated in (c). The cell observed in (a) was washed with a fresh medium after 1 h of Y-27632 incubation, and was imaged by time-lapse fluorescence microscopy. The lamellopodium located in the cell periphery disappeared and the typical central stress fibers were gradually reorganized within 1.5–2 h. The strong accumulation of GFP-{alpha}-actinin in the central portion of the cell was observed within 5 min (c; arrows in 0 : 04 min). See Supplementary Video S3. Bar, 20 µm.

 
The Rho-kinase inhibitor's effects on the GFP-{alpha}-actinin transfected cells were reversible. In order to reduce the Rho-kinase inhibitor's effects on the cells, the GFP-{alpha}-actinin transfected cells were washed with a fresh medium, which caused the reorganization of the {alpha}-actinin containing stress fibers and focal adhesions (Fig. 2c; see Supplementary Video S3). After the inhibitor was removed, the strong accumulation of GFP-{alpha}-actinin in the central portion of the cell was observed within 5 min (Fig. 2c, arrows in 0 : 04 min). The small plaque-like remnants of the focal adhesions remained localized in the center of the cell. The GFP-{alpha}-actinin containing lamellopodium decreased gradually, followed by the reorganization of the stress fibers in the central portion of the cell. As we previously mentioned, although not all of the focal adhesions were visible by GFP-{alpha}-actinin, at least some of the remaining focal adhesions were used to reorganize the stress fibers in the cell center.

In order to observe the Rho-kinase-dependent reorganization of the focal adhesions, we observed the living fibroblasts transfected with DsRed2-paxillin, a typical focal adhesion associated protein, using a time-lapse fluorescence recording system for up to 1 h with the inhibitor treatment. The major focal adhesions in the central portion of the fibroblast disappeared within 20–30 min, whereas some faint DsRed2-paxillin containing spots remained (Fig. 3; arrows; see Supplementary Video S4). This observation indicates that the Rho-kinase inhibitor selectively induces the organization of the focal adhesions that are located in the center of the cell. We also analyzed the effect of ML-7, a specific inhibitor for MLCK, using fibroblasts transfected with DsRed2-paxillin. ML-7 selectively affected the peripheral focal adhesions within 30 min. They lost the peripheral focal adhesions and then gradually became rounded. The central focal adhesions were still visible after the treatment of ML-7, thus indicating that the focal adhesions in the peripheral portion are regulated by the MLCK regulatory system and the focal adhesions in the central portion are regulated by Rho-kinase (see Supplementary Video S5).


Figure 3
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  		Figure 3  The effects of a Rho-kinase inhibitor in a living fibroblast expressing GFP-labeled paxillin. After the incubation with the Rho-kinase inhibitor, the central focal adhesions selectively disappeared (arrows). Although the number of peripheral focal adhesions decreased slightly, they persisted throughout the observation period. Bar, 20 µm. See Supplementary Video S4.

 
When the cells were incubated with Y-27632 for 1 h, then washed with a fresh medium to remove the drug, the focal adhesions gradually reorganized in the center of the cell (Fig. 4; see Supplementary Video S6). Some remnants of the focal adhesion-like structures were also enlarged and moved toward the center of the cell after the incubation with the fresh medium. The selective reorganization and/or enlargement of the focal adhesions caused by the Rho-kinase activation was observed mainly in the center of the cell.


Figure 4
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  		Figure 4  The recovery of the central focal adhesions in the cell washed after 1 h with Y-27632 treatment. The same cell in Fig. 3 was washed with a fresh medium and the time-lapse fluorescence images were recorded. After the removal of the Rho-kinase inhibitor, the cell contracted slightly and then organized newly formed central focal adhesions (arrows and arrowheads). Bar, 20 µm. See Supplementary Video S6.

 
The recovery of cells from the Rho-kinase inhibitor was also examined by fluorescence microscopy and replica electron microscopy. In the central portion of the cell, after recovery from the drug for 30 min, many small clusters of the focal adhesions as revealed by anti-vinculin antibody were visible at the central portion of the cell (Fig. 5c). However, the bundling of actin filament was still immature (Fig. 5b). The double staining with the anti-{alpha}-actinin antibody and rhodamine-labeled phalloidin also revealed that the clusters of anti-{alpha}-actinin positive focal-adhesion like structures were apparently organized at the center of the cell (Fig. 5f), but only a loosely arranged bundling of actin filaments (Fig. 5e) and its associated anti-{alpha}-actinin staining was visible (Fig. 5f). Above finding indicates that the stress fibers were still immature within 30 min after the recovery form drug. However, the ends of the loosely arranged actin filament bundles were firmly anchored to the vinculin and the {alpha}-actinin-containing focal adhesion-like structures. The replica electron microscopy, which followed the inhibitor recovery for 30 min, also revealed that although the bundling of the microfilaments was still loose (Fig. 5g; arrows), focal adhesion-like structures were apparently visible at the end of the reorganizing bundling of the microfilaments (Fig. 5g; arrowheads). These focal adhesion-like structures were firmly attached to the glass surface, reflecting an anchoring structure to form bundles of actin filaments which were subsequently observed as developed stress fibers.


Figure 5
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  		Figure 5  The recovery of central stress fibers in cells washed after 30 min of Y-27632 treatment. The recovering cell was fixed in 30 min and doubly stained with rhodamine-labeled phalloidin (b) and anti-vinculin (c). The yellow staining indicates where two colors overlap in merged images (a). The central stress fibers and focal adhesions were reorganized within 30 h after washout of the drug, but the bundling of the actin filaments were still loose within 30 min after recovery. The cells which were doubly stained with rhodamine-labeled phalloidin (e) and anti-{alpha}-actinin (f) were also indicated. The accumulation of anti-{alpha}-actinin was visible in the focal adhesions (f; arrowheads) and reorganizing loosely arranged actin filament. The replica electron microscopy of microfilaments and focal adhesion-like structures isolated on a coverslip after the recovery after 30 min from the Y-27632 treatment was also shown in (g). The small, focal adhesion-like structures were visible (g; arrowheads), along with the sparsely localized microfilaments associated with them (g; arrows). Note that the formation of bundles of microfilaments was still immature (g). Bar in (f) is 20 µm. Bar in (g) is 1 µm.

 
In the central portion of the cell, after recovery from the drug for 1 h, many focal adhesions were observed, along with the bundling formation of the actin filaments (Fig. 6a–c). Some of the stress fiber bundles were still immature, but some complete bundles of the actin filaments were observed at the central portion of the cell (Fig. 6c). The ends of the stress fibers were firmly attached to the vinculin-containing focal adhesion-like structures (Fig. 6c,f). The accumulation of anti-{alpha}-actinin staining was also observed along the phalloidin-stained structures, reflecting that the well-developed stress fibers were organized at the central portion of the cell at this time (Fig. 6e). The time-lapse recording of the living fibroblasts transfected doubly with DsRed2-paxillin and GFP-actin revealed that the reorganization of the focal adhesions preceded the bundling of the actin filament (see Supplementary Video S7a and S7b for doubly transfected with GFP-actin and DsRed2-paxillin). The replica electron microscopy also revealed the bundled formation of microfilaments in the center of the cell after recovery from Y-27632 for 1 h (Fig. 6g). Both focal adhesion-like structures (Fig. 6; arrowheads) and reorganized bundles of microfilaments (Fig. 6g; arrows) were firmly attached to the glass surface.


Figure 6
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  		Figure 6  The recovery of central stress fibers in cells washed after 1 h of Y-27632 treatment. The recovering cell was fixed in 60 min and doubly stained with rhodamine-labeled phalloidin (b) and anti-vinculin (c). The cells which were doubly stained with anti-{alpha}-actinin (e) and anti-vinculin (f) were also indicated. The yellow staining indicates where two colors overlap in merged images (a and d). Both the central stress fibers and focal adhesions were reorganized within 1 h after washout of the drug. A full recovery was observed 1.5–2 h after washing. The replica electron microscopy of the central stress fibers and focal adhesions isolated on a coverslip after the recovery from the Y-27632 treatment for 1 h were shown in (g). Well developed microfilament bundles (g; arrows) were observed within 60 min after the washout of Y-27632 along with the structure of focal adhesions (g; arrowheads). High power view of the focal adhesion structure was indicated (g; Inset). Bar in (f) is 20 µm. Bar in (g) is 1 µm.

 
We wondered whether or not the Rho-kinase could independently regulate focal adhesion formation with the organization of stress fibers. In order to examine the Rho-kinase regulation of the focal adhesion independently with stress fibers, we treated the living cells together with the Rho-kinase inhibitor and selective myosin II inhibitor, Blebbistatin or BDM (Fig. 7). When the cells were treated together with Y-27632 and Blebbistatin for 1 h, both the peripheral and the central stress fibers disappeared and the cells showed a rounded morphology. The anti-paxillin positive adhesion plaque-like structures were also independently visible with phalloidin staining at the basal portion of the cell (Fig. 7a–c). When the cells were washed by a medium containing Blebbistatin in order to remove the Y-27632 effect, the accumulations of the anti-paxillin positive clusters increased in number and in size at the cell center (Fig. 7d–f). The treatment of BDM in the replacement of Blebbistatin, another Myosin II inhibitor, also showed a similar result (data not shown). The above observations reflect that the Rho-kinase-dependent reorganization of the focal adhesions was independently regulated with the formation of the stress fibers.


Figure 7
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  		Figure 7  The effects of the myosin II inhibitor (Blebbistatin) on the Rho-kinase dependent-organization of the focal adhesions. The cells treated with both Y-27632 and Blebbistatin were stained with rhodamine-phalloidin (b) and an anti-paxillin antibody (c). The overlapped images were indicated in (a). The treatment of both inhibitors for 1 h significantly reduces both the central and the peripheral stress fibers (b). The accumulation of anti-paxillin staining is reduced and randomly localized at the basal portion of the cell (c; arrowheads). After washing with a medium containing only Blebbistatin in order to reduce the Y-27632 effect, the reorganization of the stress fibers was still not visible (e), but the staining of the anti-paxillin positive plaque-like structures was enlarged in size and in member (f; arrowheads). The overlapped images were indicated in (d). Bar, 20 µm.

 
Phosphorylation of MLC during Rho-kinase-dependent organization of stress fibers

It is generally known that Rho-kinase phosphorylates MLC. The effect of Rho-kinase in the phosphorylation of MLC was examined by fluorescence microscopy with a phosphorylated-MLC specific antibody. This observation ascertained the phosphorylation level of MLC during the Rho-kinase-dependent organization of the stress fibers. Figure 8a is a control fibroblast stained with anti-phosphorylated MLC. Both the peripheral and central stress fibers were intensely stained by the antibody, reflecting that the stress fibers generate a contractile force which is generated by the actomyosin system throughout the cell under normal conditions. After the incubation with Y-27632 for up to 1 h, the staining of the anti-phosphorylated-MLC in the center of the cell completely disappeared. However, in the cell periphery, the staining of anti-phosphorylated MLC was reduced but still visible (Fig. 8b,c; arrows). When the cells were treated with the inhibitor for 1 h and then washed with a fresh medium in order to remove the drug, the typical staining of the anti-phosphorylated MLC of the newly generated stress fibers located at the central portion were again visible within 30 min (Fig. 8d). The phosphorylation level of MLC recovered within 60 min after recovery from the effects of the inhibitor (Fig. 8e,f). This observation indicates that MLC phosphorylation is essential for the Rho-kinase-dependent reorganization of the stress fibers in the center of the cell.


Figure 8
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  		Figure 8  The distribution of phosphorylated-MLC in the reorganized stress fibers washed after 1 h of Y-27632 treatment. The control fibroblast is indicated in (a). The drug-treated cells (b: Y-27632 for 1 h) were washed and fixed in 0 : 00 min (b), 0 : 15 (c), 0 : 30 (d), 1 : 00 (e), or 1 : 30 (f) and then stained with anti-phosphorylated myosin, and the typical staining patterns are shown. The arrows indicate the phosphorylated MLC positive peripheral stress fibers. Note that the phosphorylated MLC positive central stress fibers are visible within 0 : 30 after Rho-kinase activation. The bright staining along the nucleus is indicative of a nonspecific binding of a secondary antibody. Bar, 20 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The organization of stress fibers is strongly dependent on the phosphorylation of MLC. The current study demonstrates that the phosphorylation level of MLC revealed by anti-phosphorylated myosin staining of a fixed fibroblast is increased along the reorganized stress fibers. After incubation with the Rho-kinase inhibitor for up to 1 h, the phosphorylation level of MLC was retained along the peripheral stress fibers, although the phosphorylation level was slightly decreased. This finding strongly suggests that the contractile activity is retained in the cell periphery even if the cells are treated with Rho-kinase inhibitors. Rho-kinase activity is selectively influenced by the formation of stress fibers that are located in center of the cell. Moreover, the phosphorylation of MLC caused by Rho-kinase activity is needed to maintain the central stress fibers. This observation is consistent with the idea that Rho-kinase mediates the organization of stress fibers that are located in the center of the cell (Totsukawa et al. 2000; Katoh et al. 2001a,b). The phosphorylation of MLC in the actomyosin systems generates a contractile force, thus it can be emphasized that the Rho-kinase-sensitive central stress fibers and their associated focal adhesions play a crucial role in the maintenance of cell shape and tension.

How and when does the Rho-kinase-dependent organization of stress fibers and focal adhesions occur? In this study, we revealed that the Rho-kinase dependent reorganization of the stress fibers occurs in a small, focal adhesion-like structure in the cell center, and that the actin filament accumulation and bundling occurs around the focal adhesion-like structure within 15–30 min. During this period, the phosphorylation level of MLC along the bundling gradually increases, and the accumulation of {alpha}-actinin simultaneously occurs. This observation strongly suggests that the Rho-kinase affects not only the organization of the stress fibers, but that it also influences the component of the focal adhesion associated proteins. The organization of the focal adhesion-like structure seems to precede the organization of the stress fibers. So far, there has been no apparent evidence regarding which appeared first, stress fibers or focal adhesions. Our present experiments suggest that the focal adhesion-like structures are organized earlier than the bundling of the actin-containing microfilaments. This means that, as far as Rho-kinase-dependent organization, the focal adhesions are organized earlier than the stress fibers. Moreover, the Rho-kinase-dependent organization of the focal adhesions seems to occur independently of the formation of the stress fibers. The functional mechanism of Rho-kinase for the organization of the focal adhesions remains unclear. However, it is presumable that the Rho-kinase directly or indirectly influences the accumulation of the focal adhesion associated proteins during the organization of the focal adhesions.

We recently developed a method for the mass isolation of stress fibers from cultured cells (Katoh et al. 1998, 2000). Using isolated stress fibers, we have already confirmed that two independent ways, a calcium ion-dependent calmodulin/MLCK system and a calcium ion-independent Rho-kinase system, regulate the stress fiber contraction systems, at least in cultured cells. Moreover, the calmodulin/MLCK-dependent system mainly regulates the peripheral stress fiber contraction, while the Rho-kinase-dependent system mainly regulates the central stress fiber contraction (Katoh et al. 2001a). Both peripheral and central stress fibers contain both the calmodulin/MLCK and Rho-kinase-dependent contraction systems. A sophisticated regulation system for the contraction of stress fibers should exist in two types of stress fibers. The selective mechanisms of the Rho-kinase for central stress fiber regulation are still unknown. Further studies will be necessary in order to analyze the position-specific effects of the Rho-kinase for the organization of the stress fibers. The proposed model for the dual regulation of stress fiber organization in both the peripheral and central portions of the cell are shown in Fig. 9.


Figure 9
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  		Figure 9  The proposed model for the dual regulation of stress fiber organization in both the peripheral and central portion of the cell. Both MLCK and Rho-kinase individually regulate two types of stress fibers, peripheral and central stress fibers, respectively. The depicted pathway coordinates the organization of certain types of stress fibers and systematically regulates cell contraction.

 
{alpha}-Actinin cross-links actin filaments into actin filament bundles. In combination with {alpha}-actinin and myosin II, the actin filaments develop well-organized contractile machinery within certain types of cells. {alpha}-Actinin seems to play an important role in the organization of the focal adhesions. In vitro analyses have demonstrated that {alpha}-actinin makes a direct connection between stress fibers and transmembrane proteins called integrins. It is also well known that {alpha}-actinin directly associates between the actin filament and the focal adhesion structural protein named vinculin. There is no direct evidence that Rho-kinase regulates the {alpha}-actinin distribution in both the stress fibers and focal adhesions. However, the accumulation of {alpha}-actinin in both the stress fiber and focal adhesions was observed after the Rho-kinase activation in the center of the cell. It is conceivable that Rho-kinase affects the phosphorylation of MLC, but that it also regulates the localization of the actin filament bundling proteins, such as {alpha}-actinin. Rho-kinase thus affects not only the phosphorylation of MLC, but it also regulates cytoskeletal proteins. For example, Rho-kinase regulates the activity of adducing and the ERM family (ezrin, radixin and moesin) proteins (Fukata et al. 1998; Kimura et al. 1998). However, the mechanisms of the Rho-kinase-dependent accumulation of focal adhesion associated proteins for the organization of focal adhesions remain unknown. However, it is presumable that the specific target of Rho-kinase is not only the component of stress fibers, but also the component of focal adhesion associated proteins. {alpha}-Actinin is one candidate for this type of Rho-kinase target. This could cause the Rho-kinase-dependent organization of the focal adhesion-like structures, which are observed in this study, independent of the localization of the actin filament bundles. The control mechanism for the regulation of the focal adhesions by Rho-kinase is a very interesting issue that needs to be resolved.


   Experimental procedures
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell culture

The fibroblasts (FS-133, WI-38 and NIH 3T3) were cultured using a 1 : 1 mixture of Dulbecco's modified Eagle medium and a nutrient mixture F-12 (Gibco, Grand Island, NY), pH 7.4 containing 50 units/mL penicillin, 50 µg/mL streptomycin and 10% fetal bovine serum (Salmond Smith Biolab, New Zealand). The cells were maintained at 37 °C in a humidified, 5% CO2 atmosphere.

Antibodies

The monoclonal anti-vinculin antibody (Sigma, St. Louis, MO), anti-paxillin (Zymed) and anti-{alpha}-actinin (Abcam, Tokyo, Japan) were purchased. A polyclonal anti-phosphorylated myosin regulatory light chain specific antibody was also purchased (Santa Cruz Biotechnology, Santa Cruz, CA). Rhodamine-labeled phalloidin was also purchased for actin filament staining (Molecular Probes, Eugene, OR).

Inhibitors

The Rho-kinase inhibitor, Y-27632, was purchased from Tocris (Ellisville, MO) (Uehata et al. 1997). The Rho-kinase inhibitor, HA1077, was purchased from Upstate Biotechnology (Lake Placid, NY; Takayasu et al. 1986; Uehata et al. 1997). Myosin disrupting regent (S)-(–)-Blebbistatin (Cosmo Bio, Tokyo, Japan) and inhibitor of actin-myosin interactions, 2,3-butanedione 2-monoxime (BDM; Sigma) were also purchased.

Replica electron microscopy of isolated stress fibers

The stress fibers were isolated using a modified method, as described in our previous report (Katoh et al. 1998, 2000). Briefly, FS-133 or WI-38 cells cultured for 3–4 days on a coverslip (15 x 15 mm) were incubated with the appropriate inhibitor for an exact time and then quickly washed in an ice-chilled, phosphate-buffered saline (PBS). Next, they were extracted for 5 min with gentle agitation in a low ionic strength solution consisting of 2.5 mM triethanolamine (2, 2', 2'-Nitrilotriethanol, TEA) (Wako, Osaka, Japan), 20 µg/mL Trasylol (Bayer, Leverkusen, Germany), 1 µg/mL leupeptin (Peptide Institute, Osaka, Japan) and 1 µg/mL pepstatin (Peptide Institute), pH 8.2 at 4 °C. At this stage, most of the cells had lost their dorsal side and nuclei, but some had retained these parts of the cell, which then were removed by gentle shearing with a stream of extraction buffer under a phase contrast microscope. The stress fiber and its associated focal adhesion attached to the glass surface were gently washed by a Triton X-100 based extraction buffer [0.05% Triton X-100 (Wako), 20 µg/mL Trasylol, 1 µg/mL leupeptin and 1 µg/mL pepstatin in PBS, pH 7.4] for 3 min. The stress fibers on the coverslips were fixed with 1% glutaraldehyde, dehydrated through graded concentrations of ethanol (50%, 65%, 75%, 85%, 95% and 99%) and 2-methyl-2-propanol (99%), and then freeze-dried. The samples were rotary shadowed with platinum/carbon at an angle of 45° on a JFD-9000 freeze fracture apparatus (JEOL, Tokyo, Japan). The replicas were detached from the glass by a brief treatment with 10% hydrofluoric acid and mounted on Formvar film-covered grids. The samples were examined under a JEOL 2000EX electron microscope at an accelerating voltage of 80 kV.

Immunofluorescence microscopy

The cells were fixed with a 1% paraformaldehyde in PBS for 30–60 min and treated with a 10% normal goat serum for 1 h at room temperature. They were then stained with an anti-vinculin antibody (1 : 400), anti-paxillin (1 : 100) anti-{alpha}-actinin (1 : 50) or anti-phosphorylated MLC antibody (1 : 100) for 60 min. After being washed in PBS for 20 min, the samples were incubated with fluorescein- (Cappel, Durham, NC), labeled goat anti-rabbit or anti-mouse IgG. Some of the specimens were double-stained with one of the antibodies (and the appropriate secondary antibody) and rhodamine-labeled phalloidin. The specimens were then observed by using a confocal laser scanning microscopy (Olympus Fluoview FV-1000) with an apochromat 60x (N.A. 1.4, oil) objective lens.

GFP or DsRed2-labeled protein expression and inhibitor experiments

The cultured cells (FS-133 or NIH 3T3) were transfected with the pEGFP-actin vector which was obtained from Clontech (Palo Alto, CA), pEGFP-{alpha}-actinin kindly provided by Dr C.A. Otey, The University of North Carolina (Edlund et al. 2001) using the TfxTM.-50 Regents (Promega, Madison, WI). Cells were also transfected with pDsRed2-paxillin, pBabe-DsRed2-paxillin or pBabe-GFP-paxillin kindly provided by Dr H. Sabe, Osaka Bioscience Institute, Osaka, Japan (Mazaki et al. 1998; Nakamura et al. 2000). Some of the cells were doubly transfected with pEGFP-actin and pDSRed2-paxillin vector. The transfected cells were cultured as described above. For the time-lapse microscopy, the GFP-actin transfected cells were plated on a glass-bottom culture dish (5 cm in diameter) and placed on a temperature-controlled stage at 37 °C (Olympus, Tokyo, Japan).

For the inhibitor experiments, the transfected or intact cells were treated for 60 min with Y-27632 (10 µM) or HA1077 (30 µM). Each of these inhibitors was added to the culture medium. The samples were then observed under an inverted epi-fluorescence microscope (Olympus IX-71 with a Plan apochromat objective lens 60x, N. A. 1.4, Olympus, Tokyo, Japan) or a total reflection fluorescence microscopy (TRIFM; Olympus) and the time-lapse images were recorded. Some of the samples were fixed with paraformaldehyde and stained simultaneously with anti-vinculin (Sigma) and rhodamine-labeled phalloidin as described above. The recovery experiments were performed by treating cells with one of these inhibitors for 1 h and then washing them with a fresh medium, and the process of recovery was recorded as described above. Some of the cells were treated together with Y-27632 (10 µM) and Blebbistatin (100 µM), specific myosin II inhibitor, for 60 min. To remove the effects of Y-27632, the cells were washed with a medium containing only Blebbistatin. BDM (100 µM), another myosin II inhibitor, was also used in place of Blebbistatin.


   Acknowledgements
 
We are grateful to Dr Hisataka Sabe, of Osaka Bioscience Institute, Osaka Japan, for kindly providing a pDsRed2-labeled paxillin vector. We are also grateful to Dr Carol A Otey, of The University of North Carolina at Chapel Hill, USA, for kindly providing a pEGFP-labeled {alpha}-actinin vector. We thank Mr Hatsutaka Toyama, of KS Olympus Co. Led., for permitting us to use the TIRFM (Olympus). The work reported here was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and from the Promotion and the Mutual Aid Corporation for Private Schools of Japan.


   Footnotes
 
Communicated by: Kozo Kaibuchi

* Correspondence: E-mail: katoichi{at}jichi.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 3 October 2006
Accepted: 8 February 2007





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